Cancer control consists of three components: prevention, early detection, and treatment. Although prevention and early detection present the best opportunities to cure cancer, the majority of research focuses upon treatment.
Many cancers are treatable and some are even curable (Tannock et al., 1987). However, treatment can be toxic not only to tumor cells, but also to normal cells and tissue and may be resisted by tumorous cells. To overcome these problems, researchers have sought to exploit differences between normal and tumorous cells and tissue.
First Difference--One difference between normal and tumorous cells is the amount of oxygen in the cells. Many tumorous cells of rodents and humans are oxygen deficient and are "hypoxic" (Moulder et al, 1987; Vaupel et al, 1989) (in contrast to oxygenated cells which are "oxic"). These hypoxic cells limit the cure rate of standard radiotherapy (Disch et al., 1983; Gatenby et al., 1988) and possibly some anticancer drugs (Tannock et al; 1987; Sartorelli et al., 1988). It has been shown that hypoxic cells are resistant to radiotherapeutics and chemotherapeutics (Siemann, 1992; Hill, 1987; Bremner, 1990; Workman, 1992; Coleman, 1988; Workman, 1993).
Subpopulations of cells in solid tumors, such as those of the breast, colon, brain, head and neck, are hypoxic (Rockwell, 1983). In the 1970's, hypoxia in solid tumors was suspected and, in the 1980's, was confirmed (Siemann, 1992; Brown, 1979; Chaplin, 1987; Brown, 1979). Drug resistance in solid tumors may be caused by hypoxia (Rice et al., 1988). It has been shown that increasing the oxygen levels in experimental tumors decreases resistance to radiotherapy while decreasing the oxygen levels increases resistance to radiotherapy (Siemann, 1992; Bremner et al., 1990; Workman, 1993; Olive et al., 1992).
Hypoxic cells of solid tumors are resistant to chemotherapy for a number of reasons: lacking a normal cell growth cycle, they are insensitive to cycle-specific agents (Bremner et al., 1990); their location makes them poorly accessible to cytotoxic drugs; and their lack of oxygen affects the activity of drugs which have oxygen-dependent processes (Bremner et al., 1990).
Tumor cells become hypoxic as they multiply. Those cells close to a blood supply receive the necessary oxygen for proliferation. As the cells multiply and the tumor enlarges, the rapid cell growth exceeds the vascular development reducing the tumor's supply of oxygen (Vaupel et al; 1989; Siemann; 1992; Brown, 1979). Consumption of oxygen by the cells near the supply limits the amount of oxygen available to cells away from the supply (Vaupel et al., 1989). This results in varying degrees of hypoxia.
Tumor cells adjacent a blood supply are non-hypoxic, while those more than 120-150 mm away from a blood supply are chronically hypoxic (Vaupel et al., 1989). Between fully oxygenated cells and fully hypoxic cells are cells with varying degrees of hypoxia (Vaupel et al., 1989). Intermittent vascular occlusion or collapse results in acutely hypoxic cells.
Second Difference--Another difference between normal and tumorous tissue is related to this lack of oxygen. Reductive metabolic processes may be more prevalent in the hypoxic environment of solid tumors (Workman et al., 1993). Reductive enzymes reduce functional groups (such as N-oxides) having a potential to be reduced. Nitro compounds are reduced to amino derivatives and quinones are reduced to hydroquinones by enzymes such as DT-diaphorase, cytochrome P.sub.450, cytochrome P.sub.450 reductase and xanthine oxidase (Walton et al., 1989). It has recently been shown that DT-diaphorase levels tend to be elevated in human tumor samples from lung, liver, colon and breast cancers (Workman, 1994).
These two differences between normal and tumorous cells has led to the development of bioreductive antitumor drugs. These are drugs which exploit (1) the hypoxic nature, and (2) the reductive nature, of tumorous cells. These drugs are nontoxic and inactive until they are reduced by hypoxic cells thereby becoming toxic and active, cytotoxic agents (Workman, 1992).
A number of N-oxides have been examined recently for this bioreductive activity. One is the N-oxide derivative of 1,4-bis-{[2-(dimethyl-amino)ethyl]amino}. 5,8-dihyroxyanthracene-9, 10-dione (AQ4N). This N-oxide is more toxic in vivo under conditions that promote transient hypoxia or which diminish the oxic tumor fraction (Patterson, 1993). Others are the mono-N-oxides of fused pyrazines, the lead compound of which is RB 90740. The N-oxide function is essential for the differential cytotoxic properties of these agents (Adams, 1992). Another is the aliphatic N-oxide of nitacrine, SN 24030. It has an exceptionally high selectivity for hypoxic cells (approximately 1500 fold) and an improved ability to diffuse into the extravascular compartment of tumors (Wilson et al., 1992). The N-oxide itself does not provide a reactive species but the reduction of this functional group unmasks an agent with cytotoxic potential
However, so far, none of these N-oxides has been found to have clinical activity and to lack toxicity to normal cells and tissue.
One N-oxide derivative which has been studied with little success to date as an anti-tumor agent is the N-oxide derivative of chlorambucil (also known as a nitrogen mustard derivative). Chlorambucil is toxic to tumorous cells (McLean et al., 1979). Chlorambucil acts as an anti-tumor agent by cross-linking (or alkylating) DNA, preventing DNA from replicating and cells from growing. Chlorambucil has this effect in both tumorous and normal cells (Powis et al, 1991).
Previous studies on corresponding compounds have indicated potential for anticancer activity, but no selectivity under hypoxia. Japanese Patent No. 5073 (Ishidate and Sakurai), issued on Jul. 23, 1955, describes a method of manufacturing a related derivative of N-methyl nitrogen mustard N-oxide HCI (known as nitromin) namely, N-chloroethoxy N-chloroethyl N-methyl amine. The patent claims that this agent, the rearranged derivative of nitromin, is useful in treating cancer, but does not describe how to use the derivative to treat cancer, the relevance of hypoxia or whether this derivative converts to nitromin in vivo. Indeed, Ishidate later reported that nitromin was more stable but less reactive than N-methyl nitrogen mustard under the conditions tested (Ishidate et al. 1960). Ishidate showed that a lethal dose of nitromin which killed 50% of experimental animal was 50 times less toxic than its corresponding nitrogen mustard. Nitromin was found to be readily absorbed after oral administration and excreted rapidly, largely unchanged in the urine. This study, however, did not determine (1) the contribution of reductive enzymes to the in vivo cytoxicity of nitromin, (2) whether nitromin is stable in hypoxic and oxic cells, (3) whether nitromin is toxic in cells having varying degrees of hypoxia, and non-toxic in oxic cells at corresponding concentrations.
A recent study of nitromin has shown that reduction by cyt P.sub.450 reductase regenerates the potent bifunctional alkylating species N-methyl bis(B-chloroethyl)amine (White et al. 1992).
A number of persons have also recently studied the N-oxide derivative of chlorambucil to determine whether this agent would provide selective toxicity to hypoxic tumor cells. A study has reported that the N-oxide of chlorambucil is ineffective as an anti-tumor agent because this derivative is not preferentially toxic under hypoxia (Mann et al., 1991 ). A very recent paper again reported that the N-oxide of chlorambucil shows no enhancement of hypoxic selectivity beyond the value for chlorambucil (Denny et al. 1994).
Neither of these studies examined the effect of the N-oxide of chlorambucil under bioreductive conditions which might mimic conditions in vivo. The 1991 and 1994 studies (Mann et al. 1991; Denny et al. 1994) examined the N-oxide in vitro using cell lines which lack the levels of reductive enzymes which would be able to reduce the derivative. In addition, the 1960 study (Ishidate et al., 1960) examined nitromin in vivo using Ascities cells, which are oxygenated. Therefore all of these studies failed to mimic the hypoxic conditions of tumourous cells in vivo.
This inventor has reported that the N-oxide derivative of chlorambucil is less cytotoxic than chlorambucil and that under hypoxic conditions its cytotoxicity and metabolism are potentiated by the presence of reducing enzymes (Kirkpatrick et al., 1994; Kirkpatrick et al., 1994). These studies have been discounted by others who were unable to demonstrate the selective toxicity of chlorambucil N-oxide under hypoxic conditions (Denny et al., 1994).
Thus, apart from this inventor's work, published papers on N-oxide derivatives of chlorambucil have maintained that such derivatives show either weak or no enhancement of cytotoxicity under hypoxic conditions. Thus, there is a need to develop N-oxide derivatives of chlorambucil which (1) are stable in hypoxic and oxic cells, (2) are toxic in cells having varying degrees of hypoxia, and (3) show little toxicity to oxic cells.
In this application, "CaNT" tumor cells means CaNT murine adenocarcinoma cells. "CHL" means chlorambucil, which is a nitrogen mustard, and its variants. "CHL-HD" means 4-[p-(N-2-chloroethoxy N-2-chloroethylamine)phenyl] butanoic acid or the hydroxylamine form of chlorambucil. "CHLN-O" means an N-oxide derivative of chlorambucil. "EMT6" cells means mouse mammary tumour cells. "HYDRAL" means hydralazine. "Hypoxic" or "hypoxia" means oxygen deprived. "NBP" means 4-(p-nitrobenzyl)pyridine (NBP). "NADPH" means nicotinamide adenine dinucteotide phosphate in reduced form. "Oxic" means oxygenated. "SF" means survival fraction. "Tumor" or "tumorous" means cancerous cells or tissue.